As shown in FIG. 2, for identification of an amino acid derivative in the last stage of Edman degradation, which is a sequence analysis method of a protein from the N terminal, protein is conventionally reacted with phenylisothiocyanate (PITC) to obtain phenylthiocarbamyl (PTC) protein, which is treated with an acid to produce 2-amino-5-thiazolinone (ATZ) amino acid derivative, which is further treated with an acid to form phenylthiohydantoin (PTH) amino acid derivative, which is detected by ultraviolet absorption spectroscopy as a known analysis method (P. Edman, Acta Chem. Scand. 10, 761 (1956)).
Further, another method is disclosed in Japanese Patent Application Laid-Open No. 61-264264, and shown herein in FIG. 3, in which an ATZ amino acid derivative is reacted with an amino compound labeled with a radioactiveisotope to form a PTC amino acid derivative, which is separated by thin-layer chromatography for detection.
Moreover, still another method is disclosed in Japanese Patent Application Laid-Open No. 63-196858 and shown in FIG. 4 herein in which an ATZ amino acid derivative is reacted with a fluorescent amino compound to form a PTC amino acid derivative, which is separated by high speed liquid chromatography for detection.
With regard to the conventional method of detecting the PTH amino acid derivative by ultraviolet absorption spectroscopy, while the detecting means is rather simple, this sequencing method is not suitable for the highly sensitive analysis which is recently demanded for treating a trace amount of protein or very trace amount of peptide.
Further, with regard to the developed highly sensitive analysis method utilizing the amino compound labeled with the radioactiveisotope, this has rather limited application in view of ill effects to the environment and, in particular, to the human body.
Moreover, with regard to the other developed highly sensitive analysis method utilizing the fluorescent amino compound, it is necessary to add a large quantity of a fluorescent reagent for maintaining a reaction rate when treating a trace amount of sample in such a liquid phase reaction. Further, it is known that the reaction yield rate decreases as a density of the sample, i.e., the ATZ derivative of protein or peptide decreases even though a large quantity of the reactive reagent is added. In such a case, the excess fluorescent amino compound which remains after the reaction with the ATZ amino acid derivative disturbs quantitative analysis of the product. Further, since the fluorescent compound is nonvolatile, it is complicated to remove the same after the reaction with the ATZ amino acid derivative.
In view of this, an object of the present invention is to provide a new method of promoting a high yield rate reaction to readily obtain the PTC amino acid derivative for highly sensitive detection.